كلونسازي ژن‌هاي bktB و phaC مؤثر در توليد بيوپليمرهاي باكتريايي تجزيه‏پذير

Cloning of bktB and phaC genes effective in production of bacterial biodegradable biopolymers

Bacterial biopolymers (Polyhydroxyalkanoates (PHAs)) are polyesters synthesised by some bacteria as an intracellular carbon and energy storage compound. These are accumulated in the cytoplasm of cells. Because of its properties as biodegradable plastics, are of commercial importance. The aim of this study was isolation and cloning of bktB and phaC genes from Ralstonia eutropha strain H16. The gene phaC encodes PHA polymerase enzyme which polymerase R-hydroxy butyryl-CoA and R-hydroxy valeryl-CoA subunits. The gene bktB encodes P ketothiolase enzyme which have an important role in the production of R-hydroxy valeryl-CoA and bacterial flexible copolymers formation. In this study bktB gene was cloned in the pGEM-T vector and transformed into competent cells that was prepared from E. coli JM107. Insertion of this fragment was confirmed by using PCR and enzyme digestion. Specific primers (with Spel and NsiI restriction enzyme sites) were designed for isolation of phaC gene.Then its entire ORF with RBS was amplified by PCR using R. eutropha genomic DNA as template. This fragment after purification from agarose gel was cloned into pGEM-bktB vector and transformed into E. coli BL21. Because of positive physical properties of copolymers in industry; the preparation of this recombinant bacterium could efficiently be used towards production of bioplastics with enhanced properties.